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Chemical Composition Antibacterial and Antifungal Activities of Flowerhead and Root Essential Oils of Santolina Chamaecyparissus L., Growing Wild in Tunisia

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Chemical Composition Antibacterial and Antifungal Activities of Flowerhead and Root Essential Oils of Santolina Chamaecyparissus L., Growing Wild in Tunisia Accepted Manuscript Chemical composition antibacterial and antifungal activities of flowerhead and root essential oils of Santolina chamaecyparissus L., growing wild in Tunisia Karima Bel Hadj Salah-Fatnassi, Faten Hassayoun, Imed Cheraif, Saba Khan, Hichem Ben Jannet, Mohamed Hammami, Mahjoub Aouni, Fethia Harzallah- Skhiri PII: S1319-562X(16)30001-8 DOI: http://dx.doi.org/10.1016/j.sjbs.2016.03.005 Reference: SJBS 692 To appear in: Saudi Journal of Biological Sciences Received Date: 27 February 2016 Revised Date: 21 March 2016 Accepted Date: 22 March 2016 Please cite this article as: K.B.H. Salah-Fatnassi, F. Hassayoun, I. Cheraif, S. Khan, H.B. Jannet, M. Hammami, M. Aouni, F. Harzallah-Skhiri, Chemical composition antibacterial and antifungal activities of flowerhead and root essential oils of Santolina chamaecyparissus L., growing wild in Tunisia, Saudi Journal of Biological Sciences (2016), doi: http://dx.doi.org/10.1016/j.sjbs.2016.03.005 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Chemical composition antibacterial and antifungal activities of flowerhead and root essential oils of Santolina chamaecyparissus L., growing wild in Tunisia Karima Bel Hadj Salah-Fatnassi a,b*, Faten Hassayoun a, Imed Cheraif c, Saba Khanb, Hichem Ben Jannet d, Mohamed Hammami c, Mahjoub Aouni a, Fethia Harzallah- Skhiri e a Laboratoire des maladies transmissibles et substances biologiquement actives LR99-ES27, Faculté de Pharmacie Monastir, Université de Monastir, Tunisie b Biological Sciences Department, Faculty of Science & Art -Rabigh, King Abdulaziz University (KAU), Saudi Arabia c Laboratoire de Biochimie, USCR de Spectrométrie de Masse, Faculté de Médecine, Université de Monastir, Tunisie d Laboratoire de chimie des substances naturelles et de synthèse organique, faculté des sciences, Université de Monastir, Tunisie e Laboratoire de Génétique, Biodiversité et Valorisation des Bioressources, Institut Supérieur de Biotechnologie, Université de Monastir, Tunisie. * Corresponding author Dr. Karima BEL HADJ SALAH Biological Sciences Department Faculty of Science & Art- Rabigh 21911 King Abdulaziz University. Saudi Arabia Tel.; +966 0507634489 E-mail address; [email protected] 1. Introduction A growing body of evidences suggested that more than three antibiotics derived from microorganisms are initiated every year (Clark, 1996, Rudi et al., 2012). Novel resources, especially plant, are also being examined. Essential oils, secondary metabolites and medicinally important compounds with or without bioactivity, have been isolated from plants belonging to the Asteraceae family: Achillea L., Anthemis L., Artemisia L., Balsamita Desf., Chrysanthemum L., Matricaria L., Tanacetum L., and Santolina Tourn (Khallouki et al., 2000). Besides the different strains, several genuses from the family Asteraceae, have been particularly shown to be antimicrobial (Paulo, 2006; Salie et al. 1996; Wetungu et al., 2014). In Mediterranean area, it has been found that the genus Santolina (Asteraceae, tribe Anthemideae) is characterized more than 10 species that are distributed widely (Derbesy et al., 1998). Usually plants of the genus Santolina cultivate in South Europe and North Africa. As a result, numerous class of this taxon have been investigated biologically and chemically and give rise to number of mono and sesquiterpenoids along with some other secondary metabolites (Barrero et al., 1998; Barrero et al., 1999; Barrero et al., 2000; Marco et al. 1993). In all these Santolina species, S. viridis Wild (South of France and North of Spain), S. pectinata Lag. (Iberian peninsula) and S. chamaecyparissus L. (Asteraceae) are the most widely spread species around the world. S. chamaecyparissus L. is synonyme of Ormenis africana (Jord. et Fourr.) Out of four species of the genus Ormenis only O. africana is vivacious (Poli et al., 1997) and others are used in important medicine (Pons Giner & Rios Canavate., 2000). Widely known shrub (S. Chamaecyparissus) with yellow inflorescences utilized in Mediterranean folk medicine because of its analgesic, bactericidal, fungicidal, vermifuge and vulnerary properties (Cuéllar et al., 1998; Grieve, 1984; Bean, 1989). Also it has been suggested that this plant is utilized in phytotherapy for numerous kinds of dermatitis (Giner et al. 1988) and also as a stimulant and a stomachic (Yoganarasimhan, 2000). Although preliminary evidence available in the literature suggested that S. chamaecyparissus has anti-inflammatory activity and anti-phospholipase A2 due to the presence of the isolated active principle nepetin present in the dichloromethane extract (Sala et al., 2000). In Tunisia, S. chamaecyparissus is the only Santolina species recorded and is used as vermifuge and emmanagogue (Le Floc’h, 1983). The components of essential oils from diverse species of Santolina have been investigated: S. oblongifolia (De Pascual et al., 1983), S. ligustica (Flamini et al., 1999), S. rosmarinifolia (Pala-Paul et al., 2001), S. canescens (Casado et al., 2001) and S. chamaecyparissus (Garg et al., 2001; Lawrence, 1997) , and all of these species produced monoterpene oils and demonstrated diverse constituents. Several volatile oils are recognized to acquire antifungal and antibacterial properties and are potentially applicable as antimycotic agents. Many scientific investigations have been conducted to evaluate the biological activities of about 60% of the essential oils possess antifungal and 35% of them exhibited antibacterial properties (Chaurasia & Vyas, 1977). A few series of studies has demonstrated the potential antimicrobial effect of essential oils from various Santolina species, i.e. Santolina corsica oil exhibited an appreciable antibacterial activity against Staphylococcus aureus (Rossi and al., 2007) sesquiterpenes from Santolina rosmarinifolia showed significant antimicrobial activity against yeasts while none of the compounds tested showed significant activity besides Gram positive and Gram negative bacteria (Barrero et al., 1999). To specifically address the constituents of an essential oil and antimicrobial properties of Tunisian S. Chamaecyparissus, we directly examined to investigate the chemical composition, antibacterial and antifungal activities of essential oils isolated by hydrodistillation from the flowerheads and roots of S. chamaecyparissus L., growing wildly in Tunisia. 2. Methodology 2.1 Plant material Plants of S. chamaecyparissus were collected at the time of flowering stage from Siliana on the mountain ‘Djebel Kesra’ located in the West Center of Tunisia at 35.81 (latitude in decimal degrees), 9.36444 (longitude in decimal degrees). The identification of plant material has been demonstrated according to the flora of Tunisia (Pottier-Alapetite, 1981) by the botanist Dr. Fethia Harzallah-Skhiri (Higher Institute of biotechnology, Monastir University, Tunisia) 2.2 Plant extraction and essential oil analysis a) Plant extraction Essential oils from the flower heads and roots of S. chamaecyparissus were found by hydrodistillation for 4 h by using a Clevenger type apparatus (Benyelles & al. 2014; Suwei & al. 2014; Okoh & al. 2010). Anhydrous sodium sulphate served to dry essential oils and stored at 4 °C until use. The yield was calculated according to the plant fresh weight. b) Essential oil analysis The identification of essential oil components was performed by GC and GC/MS. GC diagnostic were carried out on an HP5890-series II gas chromatograph (Agilent Technology, California, USA) with Flame Ionization Detectors (FID) characterised by: a fused silica capillary column, non polar HP-5 and polar HP Innowax (30 m x 0.25 mm ID, film thickness 0.25 µm). The temperature of the oven was 50 °C for 1 min, then it is programmed to achieve heating rate up to 5 to 240 °C/min and held isothermal for 4 min. Injector temperature: 250 °C, detector temperature: 280 °C; nitrogen used as a carrier gas (1.2 ml/min); and injected 0.1 ml diluted in hexane1%. The percentage of the constituents was calculated by electronic integration of FID peak areas without the use of response factor correction. Hewlett-Packard 5972 MSD system served for GC/MS analyses: HP-5 MS capillary column (30 m x0.25 mm ID, film thickness of 0.25 mm) was directly coupled to the mass spectrometry. The comparison of the components mass spectra with those in the Wiley 275 GC/MS library and of their retention indices with literature data (Adams, 2001) served to their identification. Retention indices (RI) were calculated by the retention times of a series of n-alkanes. 2.3 Antibacterial activity a) Bacterial strains The antibacterial assays were carried against four gram negative bacteria: Esherichia coli ATCC 25922, Pseudomenas aeruginosa ATCC 27853, Proteus mirabilis and Citobacter freundeii and two gram positive bacteria: Enterococcus faecalis ATCC 29212 and Staphylococcus aureus ATCC 25923 (American Type Culture Collection, Rockville, MD). Inoculums were kept in nutrient agar (Sigma) at 37°C. Bacterial suspension were prepared in Mueller Hinton Broth (Sigma) and adjusted to a108 cfu/ml for overnight incubation, b) Agar diffusion assay Antibacterial studies
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